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WO2000014276A9 - Oligonucleotides, procede et kit de detection du monocytogene de la listeria par amplification et/ou hybridation d'acide nucleique - Google Patents

Oligonucleotides, procede et kit de detection du monocytogene de la listeria par amplification et/ou hybridation d'acide nucleique

Info

Publication number
WO2000014276A9
WO2000014276A9 PCT/EP1999/006453 EP9906453W WO0014276A9 WO 2000014276 A9 WO2000014276 A9 WO 2000014276A9 EP 9906453 W EP9906453 W EP 9906453W WO 0014276 A9 WO0014276 A9 WO 0014276A9
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
seq
acid molecule
nucleotides
monocytogenes
Prior art date
Application number
PCT/EP1999/006453
Other languages
German (de)
English (en)
Other versions
WO2000014276A2 (fr
WO2000014276A3 (fr
Inventor
Pia Scheu
Alexander Gasch
Kornelia Berghof
Original Assignee
Biotecon Diagnostics Gmbh
Pia Scheu
Alexander Gasch
Kornelia Berghof
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biotecon Diagnostics Gmbh, Pia Scheu, Alexander Gasch, Kornelia Berghof filed Critical Biotecon Diagnostics Gmbh
Priority to DE59914268T priority Critical patent/DE59914268D1/de
Priority to US09/786,011 priority patent/US6797468B1/en
Priority to CA2343171A priority patent/CA2343171C/fr
Priority to JP2000569016A priority patent/JP2002524089A/ja
Priority to AU58584/99A priority patent/AU5858499A/en
Priority to EP99946095A priority patent/EP1108064B1/fr
Publication of WO2000014276A2 publication Critical patent/WO2000014276A2/fr
Publication of WO2000014276A3 publication Critical patent/WO2000014276A3/fr
Publication of WO2000014276A9 publication Critical patent/WO2000014276A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • oligonucleotides, methods and kit for the detection of Listeria monocytogenes by nucleic acid amplification and / or hybridization are provided.
  • the genus Listeria consists of the six species L. monocytogenes, L. grayi, L. innocua, L. ivanovii, L. seligen and L. welshimeri. Among these, only strains of the species L. monocytogenes are pathogenic for humans, especially for immunocompromised, elderly and newborns. The most common symptoms of listeriosis are septicemia, meningitis and miscarriages. L. monocytogenes infections are mainly caused by the ingestion of contaminated food, especially dairy products, meat, poultry and vegetables. A variety of methods for the detection of L. monocytogenes s nd known. Conventional detection methods for L.
  • monocytogenes consist of pre-enrichment and subsequent isolation of colonies on selective media (Lovett et al., J. Food Protection 50 (1987), 188-192; McClain & Lee, J. Assoc. Off. Anal. Chem. 71 (1988), 660-664). Single colonies are examined for their morphology or for their biochemical or serological properties. An analysis can take up to 6-8 days to complete.
  • Detection can be carried out by direct hybridization of probes to germ-specific DNA or RNA (see, for example, Datta, AR et al., Appl. Environ. Microbiol. 53 (1987), 2256-2259).
  • the disadvantage of such methods is the low sensitivity, since at least 10 5 -10 ° copies of the target nuclemic acid are required. This can be compensated for by combination with a multiplication of the target sequence, for example by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • germ-specific oligonucleotides are usually used, the base sequence of which is characteristic of the DNA or RNA of a specific microorganism or a group of microorganisms.
  • a hybridization to the DNA / RNA or an amplification of DNA / RNA when using these germ-specific oligonucleotides (eg as primers or probes) with the above-mentioned methods can only take place under suitable reaction conditions if the DNA / RNA of the microorganisms to be detected are present
  • the detection methods described for I. monocytogenes are mainly based on target genes that play a role in the pathogenicity of L. play monocytogenes. It is known that egg ⁇ nige ⁇ ieser genes are arranged adjacent in a virulence gene cluster on the chromosome. Since the List ⁇ olysm gene (hlyA) is initially clearly necessary for the pathogenicity of L. monocytogenes was recognized (Cossart, P. et al., Infect. Immun. 57 (1989), 3629-3636), most genotypic detection methods are based on this gene. However, the hlyA gene also occurs with a high degree of homology in apathogenic listeria (ie in L. seeligeri and L. Ivanova).
  • the sequence of the mpl gene from L. monocytogenes is described in the EMBL database under accession number X54619 [Domann, E. et al., Infect. Immune. 59: 65-72 (1991)]. Furthermore, parts of the sequence of the mpl gene of L are. monocytogenes listed in the EMBL database under accession number X60035 [Rasmussen, 0.F. et al., Infect. Immune. 59: 3945-3951 (1991)].
  • the object of the present invention was now to develop a routine-suitable detection method in which the probability of false-positive results occurring for the respective user is as low as possible even under strongly fluctuating test conditions.
  • oligonucleotide sequences are to be provided which can be used in a detection method for the metal protease gene (mpl) from L. monocytogenes.
  • oligonucleotides according to the invention can be defined as follows:
  • oligonucleotides LM1, LM 2, LM3, LM 4, LMF 1, LMF 2, LMR 1 and sequences complementary thereto in detection methods for L. monocytogenes lead to highly specific evidence.
  • the oligonucleotides LM4, LMR 1, LMF 1 and LMF 2 or the sequences complementary thereto are preferably used as probes.
  • nuclear acid molecules which are characterized in that they preferably have at least 10 successive nucleotides of their nucleotide chain
  • (d) are at least 90% homologous with a nucleic acid molecule according to claim 1.
  • the oligonucleotides according to the invention can have a length which is customary for probes or primers, in particular for a PCR reaction, they can furthermore have a length which can be prepared by amplification, in particular by means of a PCR reaction, and they can preferably have 10 to 250 bases and in particular 15 to 30 bases long.
  • Suitable as germ-specific oligonucleotides according to the invention for the detection of L. monocytogenes are therefore nucleic acids, preferably 10 to 250 bases and in particular 15 to 30 bases long, which are at least in a 10 base long sequence with the specified sequences LM 1, LM 2, LM 3, LM 4, LMF 1, LMF 2 and LMR 1 or the complementary sequences u. Smaller deviations (1 to 2 bases) in this 10 base long sequence are possible without the specified specificity being lost during the amplification and / or hybridization. The person skilled in the art is aware that in the event of such minor deviations, the reaction conditions must be changed accordingly; see. for example, T. Ma iatis, Molecular Clonmg, editor G. Samorook & EF Fritsch, Cold Spring Harbor Laboratory Press, 1989.
  • nucleic acids preferably genomic DNA
  • nuclear acid hybridization can then, using the germ-specific oligonucleotides according to the invention as sonoe, directly detect germ-specific nucleic acid sequences in the sample to be examined.
  • Various methods known to those skilled in the art are suitable for this, such as e.g. "Southern blot" or "dot blot”.
  • an indirect detection method is preferred, above all because of the higher sensitivity, in which the sought-after and released DNA / RNA sequences as described above are first used using the abovementioned.
  • Methods for amplifying nucleic acids, preferably PCR, are amplified.
  • the amplification of DNA / RNA using the methods mentioned is carried out using the nuclear acid molecules according to the invention as primers. Specific amplificates are only formed in the case where DNA / RNA from L. monocytogenes is present.
  • the specificity of the detection method can be increased by a detection reaction (downstream or during the amplification reaction) using the nuclear acid molecules according to the invention as probes. The use of incompletely germ-specific oligonucleotides is also possible for this detection reaction.
  • the nucleic acid amplification can also be carried out in the presence of one or more not completely specific oligonucleotides, so that DNA / RNA of other, undetectable microorganisms can possibly also be amplified.
  • Such an amplification method is generally less specific and should therefore be secured by a detection reaction (downstream or during the amplification reaction) with one or more of the nucleic acid molecule (s) according to the invention as probe (s).
  • various methods can be used in order to detect the amplification products produced in the indirect methods. These include methods known per se, such as visualization by means of gel electrophoresis, hybridization of probes to immobilized reaction products [coupled to nylon or nitrocellulose filters ("Southern blots") or e.g. on "beads” or microtiter plates] and the hybridization of the reaction products to immobilized probes (e.g. "reverse dot blots" or "beads” or microtiter plates coupled with probes).
  • methods can be used in which one or more nucleic acid molecules according to the invention can be used as probes in the course of the PCR reaction ("online”) to detect qualitatively and quantitatively specific amplification products.
  • nucleotides of the probes or primers according to the invention can be replaced by analog building blocks (such as, for example, nucleotides which do not occur naturally with the target nucleic acid).
  • analog building blocks such as, for example, nucleotides which do not occur naturally with the target nucleic acid.
  • up to 20% of at least 10 successive nucleotides of a nucleotide chain, in particular 1 or 2 nucleotides, can be replaced by analog components known per se for probes and / or primers.
  • Indirect detection methods can also be carried out using an internally-labeled amplificate. This can e.g. via the incorporation of modified nucleoside triphosphates (e.g. coupled to digoxigenm or to fluoresce) during the amplification reaction.
  • modified nucleoside triphosphates e.g. coupled to digoxigenm or to fluoresce
  • kits according to the invention for analytical detection methods in particular for the detection of bacteria of the species Listeria monocytogenes, is provided, which contains one or more nuclear acid molecules according to the invention.
  • the nuclear acid molecules according to the invention or the corresponding kits can be used in a method for detecting the presence or absence of bacteria of the species L.
  • monocytogenes are used in a sample, the method preferably being a nucleic acid hybridization and / or a nucleic acid amplification, such as a PCR.
  • pointing bacteria from undetectable bacteria on the basis of differences in genomic DNA and / or RNA at at least one nucleotide position in the region of one of the nuclear acid molecules according to the invention.
  • Example 1 Detection of bacteria of the species L. monocytogenes with the polymerase chain reaction
  • DNA was isolated from pure cultures of the bacteria listed in Table 1 using standard methods. About 10 to 100 ng of each of these DNA preparations was then in the presence of 0.4 ⁇ M oligonucleotide LM 1 and LM 2, or LM 3 and LM 2, 200 ⁇ M dNTP's (Boehringer Mannheim), 2.5 mM MgCl 2, 16 mM (NH 4) 2 S0 4, 67 mM Tris / HCl (pH 8.8), 0.01% Tween 20 and 0.03 U / ul Taq DNA polymethyl rase (Biomaster) used in the PCR. The PCR was carried out in a Perkin Elmer 9600 thermal cycler with the following thermal profile:
  • the amplification products were separated by means of agarose gel electrophoresis and visualized by staining with ethidium bromide.
  • the expected products of 149 bp and 151 bp in length were only observed in cases where DNA from strains of the species L. monocytogenes was present.
  • the DNA separated in the gels was transferred to nylon filters using standard methods and, for checking the specificity, hybridized with the oligonucleotide LM 4 (sequence 4) labeled at the 5 'end with digoxigenin.
  • the hybridization was carried out in 5 x SSC, 2% blocking reagent, 0.1% lauroyls-arcosine, 0.02% SDS and 5 pmol / ml probe for 4 h at 60 ° C.
  • the detection was carried out according to standard methods using alkaline phosphatase conjugates (anti-digoxiginin-AP Fab fragment, from Boehringer Mannheim) in the presence of 5-bromo-4-chloro-3-indolylphosphate and 4-nitro -Blue tetrazolium chloride (from Boehringer Mannheim).
  • Table 1 Results of the PCR amplification with the oligonucleotides LM 1 and LM 2 (SEQ ID NO 1 and SEQ ID NO 2) or LM 3 and LM 2 (SEQ ID NO 3 and SEQ ID NO 2) and the following, respectively Hybridization with the oligonucleotide LM4 (SEQ ID NO 4)
  • Example 2 Online detection of bacteria of the species L. monocytogenes with the polymerase chain reaction.
  • DNA was isolated from pure cultures of the strains and isolates listed in Table 2 using standard methods. Approx. 100 fg to 100 ng of these DNA preparations were then each in the presence of 0.4 ⁇ M oligonucleotide LM 1 and LM 2, 0.2 ⁇ M LMF 1 (label: 3 'fluorescein), LMF 2 (label: 3'-fluorescein) and LMR 1 (label: 5 '-LC Red640 (Röche Diagnostics), 3'-phosphate), 200 ⁇ M dNTP's (Röche Diagnostics) 4 M MgCl 2 , 3 ⁇ g / ⁇ l BSA (Röche Diagnostics), 16 M (NH 4 ) 2 S0 4 , 67 mM Tris / HCl (pH 8.8), 0.01% Tween 20 and 0.04 U / ⁇ l Taq DNA polymerase (HTB) were used in the PCR. The PCR was carried out in a LightCycler from Röche Diagnostics
  • fluorescence signals (detection wavelength 640 nm) were only observed in the cases in which DNA from strains of the species L. monocytogenes was present.
  • Table 2 Results of the PCR amplification with the oligonucleotides LM 1 and LM 2 (Seq ID NO 1 and SEQ ID NO 2) and in each case during the amplification reaction hybridization with the oligonucleotides LMF 1, LMF 2 and LMR 1 (SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7) , monocytogenes 4 c SLCC 4925

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une ou des molécule(s) d'acide nucléique, ainsi qu'un procédé de mise en évidence rapide et sensible de la présence de bactéries de l'espèce pathogène du monocytogène de la Listeria. L'invention concerne en outre un ou des kit(s) de test permettant de mettre ledit procédé en oeuvre.
PCT/EP1999/006453 1998-09-02 1999-09-02 Oligonucleotides, procede et kit de detection du monocytogene de la listeria par amplification et/ou hybridation d'acide nucleique WO2000014276A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DE59914268T DE59914268D1 (de) 1998-09-02 1999-09-02 Oligonukleotide, verfahren und kit zur detektion von listeria monocytogenes durch nukleinsäureamplifikation und/oder -hybridisierung
US09/786,011 US6797468B1 (en) 1998-09-02 1999-09-02 Oligo nucleotides method and kit for detecting listeria monocytogenes by amplifying and/or hybridizing nucleic acids
CA2343171A CA2343171C (fr) 1998-09-02 1999-09-02 Oligonucleotides, methode et ensemble pour detecter des monocytogenes de listeria par amplification et/ou hybridation d'acide nucleique
JP2000569016A JP2002524089A (ja) 1998-09-02 1999-09-02 オリゴヌクレオチド（Ｏｌｉｇｏｎｕｋｌｅｏｔｉｄｅ）、核酸の増幅、及び／又は、−ハイブリダイゼション（−Ｈｙｂｒｉｄｉｚｉｎｇ）によるリステリア・モノチトゲネス（ＬｉｓｔｅｒｉａＭｏｎｏｃｙｔｏｇｅｎｅｓ／Ｌ．Ｍｏｎｏｃｙｔｏｇｅｎｅｓ）の検出法とそのためのキット。
AU58584/99A AU5858499A (en) 1998-09-02 1999-09-02 Oligo nucleotides, method and kit for detecting (listeria monocytogenes) by amplifying and/or hybridizing nucleic acids
EP99946095A EP1108064B1 (fr) 1998-09-02 1999-09-02 Oligonucleotides, procede et kit de detection du monocytogene de la listeria par amplification et/ou hybridation d'acide nucleique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US19840044.6 1998-09-02
DE19840044A DE19840044A1 (de) 1998-09-02 1998-09-02 Oligonukleotide, Verfahren und Kit zur Detektion von Listeria monocytogenes durch Nukleinsäureamplifikation und/oder -hybridisierung

Publications (3)

Publication Number Publication Date
WO2000014276A2 WO2000014276A2 (fr) 2000-03-16
WO2000014276A3 WO2000014276A3 (fr) 2000-05-25
WO2000014276A9 true WO2000014276A9 (fr) 2000-07-13

Family

ID=7879597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1999/006453 WO2000014276A2 (fr) 1998-09-02 1999-09-02 Oligonucleotides, procede et kit de detection du monocytogene de la listeria par amplification et/ou hybridation d'acide nucleique

Country Status (8)

Country Link
US (1) US6797468B1 (fr)
EP (1) EP1108064B1 (fr)
JP (1) JP2002524089A (fr)
AT (1) ATE357536T1 (fr)
AU (1) AU5858499A (fr)
CA (1) CA2343171C (fr)
DE (2) DE19840044A1 (fr)
WO (1) WO2000014276A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2451498A1 (fr) 2001-06-22 2003-01-03 Marshfield Clinic Procedes et oligonucleotides pour la detection de salmonella sp., e.coli o157:h7, et de listeria monocytogenes

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE68922252T2 (de) * 1988-01-13 1995-08-24 Pasteur Institut Dns-sonde für pathogenische listeria.
DE3906832A1 (de) * 1989-01-19 1990-07-26 Boehringer Mannheim Gmbh Verfahren zur bestimmung von pathogenen listeria-bakterien
EP0418346A1 (fr) * 1989-02-06 1991-03-27 Gene-Trak Systems Sondes et procedes ameliores pour la detection de listeria
AU7998491A (en) * 1990-05-25 1991-12-31 Gilead Sciences, Inc. Sequence-specific nonphotoactivated crosslinking agents which bind to the major groove of duplex dna
US5134063A (en) * 1990-07-06 1992-07-28 Biolog, Inc. Methods for detection, identification and specification of listerias
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface
DE4318450A1 (de) * 1992-06-11 1993-12-16 Merck Patent Gmbh Verfahren und Mittel zum Nachweis von Listerien
DE59310308D1 (de) * 1992-06-11 2002-11-21 Merck Patent Gmbh Verfahren und Mittel zum Nachweis von Listerien
US5922538A (en) * 1996-11-08 1999-07-13 E.I. Du Pont De Nemours And Company Genetic markers and methods for the detection of Listeria monocytogenes and Listeria spp

Also Published As

Publication number Publication date
DE59914268D1 (de) 2007-05-03
JP2002524089A (ja) 2002-08-06
DE19840044A1 (de) 2000-03-23
EP1108064B1 (fr) 2007-03-21
WO2000014276A2 (fr) 2000-03-16
CA2343171A1 (fr) 2000-03-16
ATE357536T1 (de) 2007-04-15
AU5858499A (en) 2000-03-27
WO2000014276A3 (fr) 2000-05-25
CA2343171C (fr) 2010-05-18
EP1108064A2 (fr) 2001-06-20
US6797468B1 (en) 2004-09-28

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